1. Field of the Invention
This invention relates generally to pumping systems, particularly systems used in liquid chromatography separations and analyses. Specifically this invention pertains to providing liquid chromatography systems with a solvent sourcing capability of high reliability and high precision, and with the ability to provide time-varying compositions of solvents with high fidelity to the user-requested values, with minimum delivery delay time and delay volume, even at flow rates at or below 1 microliter per minute.
2. Description of the Prior Art
The practice of high-performance liquid chromatography (HPLC) generally requires that the molecular species to be separated or analyzed be dissolved in a liquid (the mobile phase) and conveyed by that liquid through a stationary column bed which may comprise closely packed particles or a membrane or other matrix support termed the stationary phase. The stationary phase presents a large surface area which is in intimate contact with the mobile phase. Mixtures of analyte compounds, dissolved in the mobile phase, can be separated during passage through the column by processes of adsorption or retention, which act differentially on the various analyte species. The differential retention causes the analytes to elute from the column in both a time-resolved and volume-resolved manner. The eluting analytes will typically transit through an on-line detector, where quantitative and/or qualitative examination of the analytes will occur. Additionally, in preparative chromatography, the time- and volume-resolved samples may be collected as distinct fractions, and passed on to a subsequent process for further use.
The elution behavior of analyte molecules is a function of the characteristics of both the stationary and the mobile phase. To the extent that the properties of the stationary phase may remain substantially fixed throughout the analysis, variation in elution behavior is then predominantly the result of variation in the properties of the mobile phase. In the isocratic mode of chromatography, the solvent composition remains substantially constant as a function of time, and analytes in the sample will tend to elute when a prescribed mobile phase volume has transited the column. In the gradient mode of chromatography, the solvent composition is required to change as a function of time, tracking a user-defined profile; in this mode, analytes will elute in response to both the composition of solvent delivered, and to the overall or integrated volume of solvent delivered. It is further understood that the model presented above is a highly simplified one, and that there can be more complex modes, including multiple modes, of interaction between the analyte species and the stationary and mobile phases, causing behavior which deviates from this simple model.
In light of the above, the requirements imposed on HPLC solvent delivery systems are severe. HPLC pumps are typically required to deliver solvents at pressures which can range from several pounds per square inch to as much as 10,000 pounds per square inch. Across that range of delivery pressures, HPLC pumps are expected to output the mobile phase solvent at precisely controlled flow rates, in a smooth and uniform manner. In the case of gradient chromatography, or in the case of isocratic chromatography where a fixed solvent composition is blended in real time during the separation, there is the further requirement that mobile phase composition as well as flow rate be precisely and accurately controlled during delivery, despite the fact that system operating pressure may be changing very substantially during the separation, and that the compressibilities of the constituent mobile phase solvents may be quite different. Brownlee, in U.S. Pat. No. 4,347,131, teaches the use of a single syringe-type pump for each solvent composition where each syringe is of large enough volume (typically 10 to 40 milliliter internal volume) that an entire analysis can be conducted within one cylinder delivery. The entire volume is pressurized at once and maintained online for the duration of the separation, and multicomponent solvents are blended on the high-pressure or outlet side of two or more such pumps. The implementation disclosed in Brownlee suffers from the effects of differential hydraulic capacitance presented to the system at run time, as well as transient effects associated with the discontinuous or stop/start mode of operation of these syringes. The undesireable effects of hydraulic capacitance derive from the fact that, during gradient chromatography, as solvent composition changes, solvent viscosity typically changes as well. In order for the column flow rate to remain constant, the system operating pressure must change in response to the changing viscosity.
The different solvents used to produce gradient chromatography differ markedly in their compressibilities. When two or more large, captive volumes of liquids, having differing compressibilities, are subjected to a changing hydraulic pressure, they will compress or relax to differing extents. Brownlee does not disclose any means for assuring that the solvent volume sourced to the HPLC system under gradient conditions will accurately track with the syringe displacement; instead the system disclosed in Brownlee will be in error by the amount of compression or relaxation experienced in the respective captive liquid volumes. Moreover, there is no guarantee that the volume of liquid in the Brownlee syringe will be sufficient to carry out the separation.
Trisciani et. al. (U.S. Pat. No. 4,980,296) teaches the use of a "learning cycle" which determines hydraulic capacitance prior to run-time, and stores the data in a memory, to attempt to offset these effects in syringe pumps. The weakness of this approach is that a volume correction can only be performed "after the fact" in response to a change of system pressure, which means that in practice, the correction is always lagging the intended composition sent to the column.
The large errors associated with the compression or relaxation of large volumes of fluid can be minimized by the use of small volume syringe pumps that utilize multiple syringe strokes to deliver solvent through the course of a chromatographic separation. However, these pumps suffer from flow perturbations associated with the transition of fluid delivery from one syringe cycle to the next, that transition interval being termed the syringe or piston crossover.
Likuski et. al. (U.S. Pat. No. 4,919,595) teaches use of a single syringe having a high-speed refill cycle to minimize the period of no fluid delivery. Likuski et. al. employ the gradient of the internal pressure rise of the syringe to detect the onset of the next fluid delivery cycle. The controller subsequently over-delivers to approximately make up the flow deficit, and then returns the syringe speed to normal. While this approach minimizes the period of no fluid delivery from the syringe, and reduces the average flow rate error, significant system flow and pressure perturbations still result at crossover.
Barlow et. al. (U.S. Pat. No. 4,980,059) teach the use of a single motor to drive multiple syringe pumps with overlapping delivery strokes to avoid discontinuous flow. When a substantially constant delivery rate is being maintained by a single syringe, there is a significant increase in flow when an additional syringe begins its delivery. Barlow teaches reduction of the syringe drive velocity while an additional syringe is delivering. The control arrangement monitors the delivery pressure perturbation and advances or retards the instant of change of syringe drive velocity on the subsequent stroke. This still results in the system flow and pressure being perturbed prior to corrective response.
An emerging area of chromatographic separation and analysis is developing around the use of extremely narrow bore separation columns. Such columns have been termed capillary columns, that name deriving from the internal diameter of the separation column, which will typically be in the range of 0.005 millimeters to 0.500 millimeters internal diameter. Such columns may be packed with a particulate packing material, or, in the smallest diametral range, the column wall itself, or a coating applied to that wall, will be used as the stationary phase. Mobile phase flow rates for particulate-packed capillary columns having internal diameters of 0.025 millimeters to 0.500 millimeters can typically range from 1 nanoliter per minute to 10 or more microliters per minute. These figures represent an approximately thousand-fold reduction in flow rate (and therefore volume of the separation) from what is currently practiced on, for example, the 4 millimeter internal diameter columns widely commercially available at this time. HPLC systems designed around capillary columns have particular utility when the HPLC separation is to be coupled with a downstream process which does not readily tolerate large amounts of HPLC mobile phase. Examples of such processes are: (1) mass spectrometry, which requires that the sample reside in the gas phase at high vacuum conditions prior to mass analysis, (2) infra red spectroscopy, where organic solvents used for HPLC must be eliminated because they represent an interference to analyte detection in the infra red region of the electromagnetic spectrum, and (3) micro-fraction collection, which requires that the analyte be deposited in a small volume on a collection substrate, with minimum associated background contamination from the HPLC mobile phase.
Substantially the same requirements for precision and accuracy of solvent composition and flow rate delivery exist as for larger-scale chromatography, but the mechanisms for controlling the delivery must now function at one one-thousandth the volume scale. In particular, the non-idealities of a given implementation which could be dismissed at a much larger volumetric scale give rise to overwhelmingly large perturbations to a system of the scale of capillary HPLC. Heretofore the prior art has not adequately addressed the problems of continuous, smooth flow on a capillary system scale.